This proposal will define genes of unknown function (GUF) of the Gram-negative bacteria Acinetobacter baumannii (Ab) that contribute to the structure and barrier function of the cell envelope. The Gram-negative bacterial envelope is an ordered complex of macromolecules that is unique in being comprised of two membranes. The outer membrane (OM), a unique structure composed of an asymmetrical bilayer consisting of lipopolysaccharide (LPS) (outer leaflet) and glycerophospholipids (inner leaflet), forms the first line of defense against antiseptics and antimicrobials by functioning as a permeability barrier. Ab has a novel cell envelope that is relatively impermeable to antibiotics, and colistin resistant Ab have been isolated that cannot synthesize LPS from patients treated with colistin. These novel properties suggest that the study of the Ab envelope should reveal important new knowledge of clinical relevance. Our preliminary data and published studies of transcriptional profiles of the Ab LPS null mutant have led to bioinformatics analysis and selection o f a set of Ab GUF that are candidates for participation in maintenance o f a regulated permeability barrier. These GUF will be characterized by construction of mutant strains that will be analyzed for altered permeability to chemical probes. Strains with altered envelope permeability will be subjected to susceptibility testing to antimicrobial peptides and antibiotics, chemical analysis of phospholipids and lipid A, and, when appropriate, testing in animal models of disease. In an iterative process to define new GUF for characterization in further years, we will employ specific genetic screens using FACS-based cell sorting, drug, and temperature sensitivity screens to identify GUF that contribute to cell envelope structure and function. Lastly, we will integrate data from the projects of Drs. Manoil and Harwood and utilize the technological core of Dr. Bruce to define additional GUF for characterization. This proposal will test the hypothesis that regulated undiscovered structural alterations of the Gram-negative envelope encoded by GUF are a component of pathogen defense against host innate immune, antimicrobial, and disinfectant attack.